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Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.

ABSTRACT: Cocaine esterase (CocE) is known as the most efficient natural enzyme for cocaine hydrolysis. The major obstacle to the clinical application of wild-type CocE is the thermoinstability with a half-life of only ?12 min at 37 °C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of ?6 h at 37 °C is currently in clinical trial Phase II for cocaine overdose treatment. Through molecular modeling and dynamics simulation, we designed and characterized a promising new mutant of E172-173 with extra L196C/I301C mutations (denoted as enzyme E196-301) to produce cross-subunit disulfide bonds that stabilize the dimer structure. The cross-subunit disulfide bonds were confirmed by X-ray diffraction. The designed L196C/I301C mutations have not only considerably extended the in vitro half-life at 37 °C to >100 days, but also significantly improved the catalytic efficiency against cocaine by ?150%. In addition, the thermostable E196-301 can be PEGylated to significantly prolong the residence time in mice. The PEGylated E196-301 can fully protect mice from a lethal dose of cocaine (180 mg/kg, LD100) for at least 3 days, with an average protection time of ?94h. This is the longest in vivo protection of mice from the lethal dose of cocaine demonstrated within all studies using an exogenous enzyme reported so far. Hence, E196-301 may be developed to become a more valuable therapeutic enzyme for cocaine abuse treatment, and it demonstrates that a general design strategy and protocol to simultaneously improve both the stability and function are feasible for rational protein drug design.

SUBMITTER: Fang L

PROVIDER: S-EPMC4136690 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.

Fang Lei L Chow K Martin KM Hou Shurong S Xue Liu L Chen Xiabin X Rodgers David W DW Zheng Fang F Zhan Chang-Guo CG

ACS chemical biology 20140611 8

Cocaine esterase (CocE) is known as the most efficient natural enzyme for cocaine hydrolysis. The major obstacle to the clinical application of wild-type CocE is the thermoinstability with a half-life of only ∼12 min at 37 °C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of ∼6 h at 37 °C is currently in clinical trial Phase II for cocaine overdose treatment. Through molecular modeling and dynamics simulation, we designed and characte ...[more]

PMID: 24919140

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Project description:Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.

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Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.

Publications

Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.

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